The PEGylation of proteins is a conjugation process that involves the attachment of a polyethylene glycol derivative to a therapeutic protein to improve its stability and pharmacokinetics by reducing clearance rates and providing a steric shield from proteolytic enzymes and immune system recognition (Roberts, M. J. et al., Adv. Drug Delivery Rev, 54:459 (2002)). In general, the PEGylation technologies can be classified into two types, namely random and site-specific conjugations. Random PEGylations arbitrarily link the PEGylating reagent to reactive amino acids such as lysine or cysteine to afford a mixture of PEGylated products. In contrast, site-specific conjugations exploit the unambiguous reactivity of a native functionality (e.g., the N- or C-terminal groups) or an unnatural amino acid (e.g., p-acetylphenylalanine-pAcF) to control the location and number of PEG residues attached to the protein. Site specific conjugation reaction involving ketoxime formation between a PEGylating reagent and a pAcF residue is incorporated in the substrate protein via expansion of the genetic code (Liu, C. C. et al., Annu. Rev. Biochem., 79:413 (2010); Tian, F. et al., “Accelerants for the modification of non-natural amino acids and non-natural amino acid polypeptides”, U.S. Pat. No. 7,468,458 (Dec. 23, 2008)). Despite their and demonstrated utility, conjugations based on the formation of ketoximes suffer from slow rates and incomplete conversions (Crisalli, P. et al., J. Org. Chem., 78:1184 (2013)). Attempts to improve ketoxime formation include the use of excess PEGylating reagent, high temperatures, or high concentrations of toxic catalysts. These solutions, however, introduce additional steps to eliminate the excess PEGylating reagent or toxic catalyst from the product and often compromise the stability of the protein. Additionally, the old methods do not use additives, use a denaturant (urea), and/or use acetylhydrazide (AcNHNH2) as the additive. AcNHNH2 and related structures have been defined in PCT Publication No. WO 2007/056448.
What is now needed in the art are new methods to upgrade the yield and rates for the PEGylation of proteins (Relaxin and FGF21) containing a pAcF residue by examining the mechanistic principles that effect stalling and by identifying new additives that accelerate the reaction and promote high conversions at low PEG: protein molar ratios. The methods should be economical, promote higher conversions with considerably lower amounts of PEGylating reagent, promote faster reactions that circumvent the need for high reaction temperatures and elimination of genotoxic material.